Failure of sheep\p=n-\goathybrid conceptuses to develop to term in sheep\p=n-\goat chimaeras

R. A. Gustafson , G. B. Anderson, R. H. BonDurant and G. R. Sasser department of Animal Science and department of Reproduction, University of California, Davis, CA 95616-8521 USA; and iDepartment of Animal Science, University of Idaho, Moscow, ID 83843, USA

Six were established: three in sheep\p=n-\goat chimaeras, one in a sheep\p=n-\ (sheep\p=n-\goat)hybrid chimaera and two in does. Pregnancies were monitored weekly by ultra- sonography and peripheral concentrations of specific protein B (PSPB) were measured. Placental development as detected by ultrasonography appeared to be slower in hybrid-in-goat pregnancies than in hybrid-in-chimaera pregnancies, although this difference was not reflected in PSPB concentrations. Time of fetal death could not be predicted from PSPB concentrations. Chimaeras appeared to carry hybrid pregnancies longer than ewes and does usually carry hybrid pregnancies, but none was carried to term.

Introduction measured (Allen, 1975). Pregnancy specific protein (PSPB) has been identified in maternal circulation during pregnancy in The domestic sheep, Ovis arles, and domestic goat, Capra hircus, , including (Butler et al, 1982), sheep (Ruder et do not normally interbreed. Ewes usually will not conceive al, 1988), goats (Humblot et al, 1990) and deer (Wood et al, PSPB is localized in et and secreted when bred to a buck. Through the use of artificial insemination 1986). (Eckblad al, 1985) and transfer, hybrid pregnancies have been established (Reimers el al., 1985) from the binucleate cells of the tropho- blastic ectoderm. The of PSPB has not in ewes, but they are lost by 4—6 weeks of gestation (Hancock biological function(s) et al, 1968; MacLaren et al, in press). Does will conceive when been determined but has been linked to immunosuppression et mated to a ram, but pregnancy is usually lost by 8 weeks of (Dunbar al, 1990). gestation (Warwick and Berry, 1949; Hancock et al, 1968; This study was undertaken to determine the outcome of Hancock and McGovern, 1968; McGovern, 1973a; MacLaren hybrid pregnancies in sheep-goat chimaeras, using secretion of et al, in press). Occasional sheep—goat hybrids born to does PSPB and ultrasonography to monitor pregnancy. have been reported (Bunch et al, 1976; Moore et al, 1981; Tucker et al, 1989). Dent et al. (1971) that loss of proposed Materials and Methods hybrid-in-goat pregnancies can be attributed to placental failure, possibly due to a maternal immune response (Hancock et al, 1968). Sheep-goat chimaeras provide a useful model to test this Animals theory because they are tolerant to both immunologically Establishment of hybrid pregnancies was attempted in six lines (MacLaren et 1992b; Gustafson et al, 1993). component al, female sheep—goat chimaeras produced in previous experiments If failure of is due to an immune hybrid-in-goat pregnancies (Roth et al, 1989; Ruffing, 1989), a sheep-(sheep-goat)hybrid the chimaera should be of carry¬ response, sheep—goat capable chimaera (Roth et al, 1989), two Finn ewes and two and but case. Alpine ing sheep, goat hybrid pregnancies, this is not the does. The chimaeras had previously resorbed caprine and female chimaeras were shown to lambs Eight sheep-goat carry carried to term ovine pregnancies (MacLaren et al, 1992a, b). to term, but were lost from day 23 to some goat pregnancies Hybrid embryo donors consisted of one multiparous and five time after day 40 (MacLaren et al, 1992a, b). Establishment of virgin Alpine does. Intraspecific pregnancies were obtained by hybrid in chimaeras was by pregnancies sheep-goat attempted mating three white-face ewes and an Alpine doe to a fertile ram Fehilly and Willadsen (1986); however, none developed to term and buck, respectively. and it is unclear whether pregnancy was ever established. If placental function is compromised in interspecific and hybrid pregnancies, production of placental products would be Synchronization, insemination and to be affected. Such an effect has been observed with expected All animals were synchronized by use of intravaginal mares mule where reduced con¬ carrying conceptuses greatly pessaries containing 40 mg fluorogestone acetate (Chrono-gest: centrations of chorionic are equine gonadotrophin (eCG) Intervet, Boxmeer) inserted for 10 days. Lutalyse (15 mg; 'Correspondence and reprint requests. Upjohn, Kalamazoo, MI) was administered at the time of Revised manuscript received 13 May 1993. pessary removal. Does used as hybrid embryo donors were

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access superovulated with twice daily injections of FSH (FSH-P: Table 1. Results of hybrid embryo transfer to sheep—goat Shering, Kenilworth, NJ) in decreasing doses of 5.5, 4.4 and chimaeras and to ewes 3.3 mg, respectively, for 3 days beginning the day before removal. pessary Pregnancy Insemination of the donor does with 150-200 x 106 motile Animal transferred Outcome termination* ovine spermatozoa was performed by laparoscopy to one uterine horn near the bifurcation 50—54 h after pessary removal. 8702-SHC 1 < 125 For this procedure, does were sedated with diazepam (0.15 mg Pregnant Day i.V.: Schein, Port NY) and the skin and 1 morula kg-1, Washington, 1 16-cell abdominal wall were anaesthetized by local infiltration of lido- S703-C 2 morulae Not pregnant caine Inc., NJ). Semen was collected (Elkins-Sinn, Cherry Hill, by 8704-C 1 morula Not pregnant and for 300 over a electroejaculation centrifuged 20 min at # 8806-C 2 Pregnant < Day 89 discontinuous Percoli (Sigma, St Louis, MO) gradient consisting 8809-C 3 morulae Pregnant < Day 70 of 2 ml decreasing concentrations of 70%, 50% and 40% Percoli 8810-C 1 blastocyst delayed oestrusb solution diluted in 1 x basal medium (BM). The Percoli solution 1 morula consisted of 9:1 Percoll:10 x basal medium (10 x BM: 1.13 mol 8811-C 2 morulae Pregnant < Day 49 NaCl 1 0.03 mol KCI 1 0.2 mol Tris 1 0.1 mol 102-S 1 blastocyst Not pregnant " \ " \ " \ glucose 1, 103-S 2 morulae Not pregnant 0.05 mol sodium pyruvate 1~ , 0.1 mol CaCl2 I-, 500 iu penicillin G ml-1, 0.5 ng streptomycin sulfate ml-1). The sperm pellet was then washed twice with 1 x BM and the final suspen¬ aDay ultrasonography revealed a nonviable fetus or nonpregnant state. sion held at 37°C in 1 x BM supplemented with 0.5% BSA bDelayed return to oestrus, stood to be mounted by a ram on day 46. SHC: chimaera; C: chimaera; S: (Sigma) until use. sheep—hybrid sheep—goat sheep. Morula- to early blastocyst-stage hybrid embryos were col¬ lected at 5 after insemination to laparotomy days according serum to sheep PSPB was used with bovine PSPB (laboratory methods described by Anderson et al. (1981). Embryos were preparation R-37) as the labelled standard protein. The mean held in culture in Whitten's medium and (Whitten Biggers, sensitivity of the standard curve for two assays, expressed as 0.5% BSA 1% 1968) supplemented with and (v/v) antibiotic- two standard deviations from buffer control, was 156.2 pg per solution x , Grand under antimycotic (100 Gibco, Island, NY) assay tube (sample size was 200 pi per tube). Inter- and intra- St 5% dimethyl-polysiloxane (Sigma, Louis, MO) at 37°C in assay coefficients of variation were 8.05 and 5.92%, respect¬ Ovulation the C02 until transfer to recipients. in recipients ively. Serial dilutions (with virgin ewe serum) of pooled serum (chimaeras and ewes) was confirmed at laparotomy and one to of hybrid-in-goat and hybrid-in-chimaera pregnancies inhibited three were transferred to a uterine horn to a embryos ipsilateral binding of radiolabelled (I25I) bovine PSPB in a manner parallel luteum. does were established corpus Hybrid pregnancies in by to the standard curve. Serum samples from intraspecific ovine natural to a Barbados ram. mating and caprine pregnancies were previously tested (Willard et al, 1987) and also inhibited binding parallel to the standard curve. Pregnancy detection and monitoring Animals were observed daily for return to oestrus. Chimaeras Statistical analysis were housed with a vasectomized ram a wearing marking PSPB data were subjected to analysis of covariance using the harness from 15 onwards. Ewes and chimaeras that returned day SAS General Linear Models procedure (SAS, 1985). PSPB con¬ to oestrus were bred to rams to establish naturally intraspecific centrations (absolute values and increases above basal concen¬ and ovine Animals that did not show pregnancies, respectively. trations analysed separately) were used as the dependent of oestrus 26 of oestrus = 0) were signs by day (first day day variable and type of pregnancy (for example, sheep-in-sheep, examined real-time a 3.5 MHz trans- by ultrasonography using hybrid-in-chimaera) and day of pregnancy were independent abdominal twice first for detection probe weekly, pregnancy variables with day of pregnancy used as a covariance. PSPB and then until was lost or to within monitoring, pregnancy concentrations for the different types of pregnancy were com¬ 3—4 weeks of parturition. pared at day 24 using an unpaired t test. Day-24 PSBS con¬ Blood were collected from 18 to 32, samples daily days every centrations were compared because differences resulting from other from 34 to 60, twice from 62 to 89 day days weekly days different numbers of conceptuses are not detectable at this time and once weeks weekly from day 90 until 2-3 after parturition (Humblot et al, 1990). (or 1—2 weeks after pregnancy loss). Blood was collected by jugular venepuncture into 10 ml vacutainer tubes (Becton- Dickenson, Rutherford, NJ), allowed to coagulate at room Results temperature and centrifuged at 2500 g for 10 min. The serum was collected and stored at 20°C until analysis for PSPB. — Embryo transfer Radioimmunoassay Five early blastocysts, 12 morulae, one 16-cell and four degenerating hybrid embryos were recovered from six donor Serum samples were analysed for PSPB in two radio- does. Embryos were transferred to a total of seven chimaeras immunoassays as described by Willard el al. (1987). Rabbit anti- and two virgin ewes (Table 1).

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access Table 2. Results of natural matings of sheep-goat chimaeras, ewes and does

Pregnancy Animal Bred to Outcome termination

8703-C Ram Pregnant Day 145, triplet lambs3 8704-C Ram Pregnant Day 143, single lamblb 102-S Ram Pregnant Day 143, single lamb 103-S Ram Pregnant Day 146, twin lambs 204-S Ram Pregnant Day 145, single lamb 25-G Ram Pregnantc

•"Species determined by phenotype at birth. bDelivered by Caesarian section. 'After the first breeding, doe returned to oestrus 7 days later and was rebred. Conception was assumed to have occurred at the second breeding. dDay ultra- sonography revealed a nonviable fetus. C: sheep-goat chimaera; S: sheep; G: goat.

Hybrid embryo transfer pregnancies were conclusively established in four of seven chimaeras. Two of the remaining three chimaeras, 8703 and 8704, returned to oestrus on day 16, were bred naturally to a ram and conceived. The other chimaera, 8810, had a delayed return to oestrus and stood to be mounted by a vasectomized ram wearing a marking harness on day 46. Of the two ewes that received hybrid embryos, both returned to oestrus on day 16, were bred naturally to a ram and conceived. Both does that were bred to a ram conceived, and the one doe bred to a buck also conceived. Results of natural matings are summarized (Table 2).

Ultrasonography Hybrid-in-chimaera. In the five chimaeras that received hybrid embryos and did not return to oestrus, ultrasonography revealed a fluid-filled uterus in three on day 26 and four on day 29. The fifth chimaera, 8810, returned to oestrus on day 46 and at no time before that did ultrasonography reveal signs of preg¬ nancy. On day 31 in 8809 and 8811, the uterine wall appeared irregular and by day 40 placentome formation in all four chimaeras was evident as projections along the uterine wall (Fig. la). As pregnancy progressed, the number and size of the placentomes increased and they became more c-shaped (Fig. lb, c). The fetus was not always evident, but heartbeats were first detected on day 35 and were used to determine the of the fetus. In two 8702 and 8806, in which viability cases, Fig. 1. Transabdominal ultrasonogram of (a) hybrid pregnancy in the was pregnancy progressed furthest, fetal movement used to chimaera 8809 at day 40, (b) hybrid pregnancy in chimaera 8702 at day indicate viability when the heartbeat could no longer be easily 52, and (c) sheep pregnancy in chimaera 8703 at day 58. Arrows indi¬ detected. cate placentome formation. In (a) placentomes appear as projections Chimaera 8811 was the first to resorb the hybrid pregnancy. into the central lumen; (b) demonstrates development of placentomes as or ' structures and in numerous On day 40, pregnancy was evident in the form of a fluid-filled V shaped (c) well-developed uterus and placentome formation, but by day 49 all signs of placentomes are evident. pregnancy had disappeared. The fetus was never observed directly during the course of this pregnancy. The last sign of placentomes were smaller, there was less fluid and it was a viable fetus in 8809 was a heartbeat on day 40. The fetus cloudier than observed previously. Continued loss of fluid and was next seen on day 52, but neither a heartbeat nor movement shrinking placentomes persisted until day 104, when pregnancy was detected. Signs of résorption appeared on day 70; the was no longer apparent.

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access inserted into the rectum during ultrasonography. In many ani¬ mals this approach was not successful because the uterus was beyond the range of the probe, although occasional membranes could be seen if the abdominal area was manually elevated. In both chimaeras carrying ovine pregnancies, fetal heartbeat, fetal movement and c-shaped placentomes were seen by day 39 and the placentomes continued to develop in size. Both preg¬ nancies resulted in term Iambs; 8703 had triplets by natural delivery on day 145 and 8704 had a single lamb by Caesarian section after failure of the cervix to dilate during labour on day 143.

Sheep-in-sheep and goat-in-goat. In intraspecific ovine preg¬ nancies, revealed an uterine wall, Fig. 2. Hybrid fetus obtained by Caesarian section from chimaera 8702 ultrasonography irregular fetuses and fetal heartbeat on 30—33. The on day 126. The fetus weighed 1.53 kg upon removal and had a days placentomes continued to and had obvious crown-rump length of 32 cm. develop an c-shape by day 42. Ewes 204 and 102 delivered single lambs on day 145 and 143, respectively, and ewe 103 delivered twin lambs on day 146. Chimaera 8806 had a live fetus on 70. On 89 the day day In the intraspecific caprine pregnancy an irregular uterine fetus was observed, but no movement was detected. The female wall was first detected on day 37 and the c-shape to the was marked by a vasectomized ram wearing a marking harness placentomes became apparent at about day 40 with obvious on and on 104 no of were detected day 99 day signs pregnancy by day 51. Doe 30 gave birth to quadruplets on with ultrasonography. day 149. The hybrid pregnancy in the sheep-hybrid chimaera, 8702, was carried the longest. Fetal movement was last detected on day 89. By day 104 the amount of fluid had increased Pregnancy specific protein dramatically, leading to a diagnosis of a hydroamnious possible three in condition. The abdominal measurements of the chimaera were Hybrid-in-chimaera. The hybrid pregnancies sheep- chimaeras and had lower taken twice weekly and started to decrease at about day 120. goat (8806, 8809 8811) concentrations of PSPB than did the hybrid in the Since ultrasonography on day 125 revealed neither fetal heart¬ pregnancy sheep-hybrid chimaera (8702, 3). One chimaera, 8810, with beat nor movement, the fetus was removed by Caesarian section Fig. delayed return to oestrus had very low concentrations ml-1) on day 126. Both the fetus and placenta were necrotic and (<1.9ng of PSPB or fail¬ judged to have been dead for about 5 days. The fetus had indicating non-pregnancy very early pregnancy ure. All four confirmed showed the same bulldog features to its face and its abdomen was distended with hybrid pregnancies rate of increase in PSPB concentrations to day 30, after fluid. It weighed 1.53 kg and had a crown-rump length of up which there was individual variation. In three animals (8702, 32 cm (Fig. 2). The allantochorion contained 55—57 cotyledons. 8809 and 8811), the decline of PSPB occurred at the estimated In a previous term ovine pregnancy, placental membranes from time of fetal death. In contrast, chimaera 8806 had an increase in this female contained 73 cotyledons (MacLaren et al, 1992a). PSPB concentration at the estimated time of fetal death. Hybrid-in-goat. A fluid-filled uterus was first detected in doe The concentrations of PSPB in the two 4038 on 33. By 39 a fetal heartbeat, the of Hybrid-in-goat. day day beginnings varied placentome formation and excessive accumulation of placental hybrid-in-goat pregnancies greatly (Fig. 4a). Doe 4038 had concentrations than did doe 25 to 32 and fluid were observed. On day 40 a fetal heartbeat could higher up day then the concentrations in doe 25 increased those of no longer be detected and observations on day 46 and 49 beyond 4038. In the of doe 25, PSPB concentrations declined revealed that the uterine contents were shrinking and becom¬ pregnancy at the estimated time of fetal death. In 4038 the decline ing increasingly dense. Pregnancy was no longer detectable on day 51. occurred before fetal death and there was a slight increase after the was resorbed and no detectable Doe 25 was bred to a ram and then rebred 7 days later owing pregnancy longer by This increase was also seen in chimaera 8806. to a short oestrous cycle. It was assumed that conception ultrasonography. occurred at the second breeding. Pregnancy was first detected 8703 and had on day 27 and a heartbeat on day 41. Placental development Sheep-in-chimaera. Chimaeras 8704 unusually did not progress beyond irregularities along the uterine wall, high basal concentrations (day 0) of PSPB, 19.52 and 8.27 ng first detected on day 41. Last signs of fetal viability were ml-1, respectively. Both animals had increased concentrations observed on day 44. The fetus was detected on days 50 and 57, of PSPB until approximately day 60 and then concentrations but no heartbeat or movement was observed and all signs of declined. In 8703, PSPB began to rise again at about day 105 pregnancy disappeared by day 62. Excessive accumulation of but in 8704 concentrations remained low (Fig. 4b). placental fluids was not apparent. Sheep-in-sheep and goat-in-goat. The profile of PSPB in Sheep-in-chimaera. Pregnancy was first detected in chimaeras intraspecific ovine pregnancies showed two peaks, although the 8703 and 8704 on day 26 by using a 5 MHz linear probe timing of these peaks varied among individuals (Fig. 4c). In two

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access 120 130 140 150

Fig. 3. Pregnancy specific protein (PSPB) concentrations in peripheral blood of hybrid- in-chimaera pregnancies. Arrows indicate when a nonviable fetus or nonpregnant state was detected by ultrasonography. Chimaera 8810 was not confirmed pregnant. Values for individual animals: (·) 8806; (O) 8809; ( ) 8810; (D) 8811; (A) 8702.

Fig. 4. Pregnancy specific protein (PSPB) concentrations in peripheral blood of (a) hybrid-in-goat, (·) 25, (O) 4038; (b) sheep-in- chimaera, (·) 8703, (O) 8704; (c) sheep-in-sheep, (·) 204, (O) 102, ( ) 103; and (d) goat-in-goat pregnancies. Arrows in (a) indicate when a nonviable fetus was detected by ultrasonography. animals, ewes 102 and 103, the first and second peaks occurred Overall The rate of change of PSPB from days 18-30 did at approximately day 60 and day 130, respectively. In the third not differ among animals (slope = 0.4501, > 0.05). Absolute animal, ewe 204, the peaks occurred at approximately day 90 concentrations of PSPB (P < 0.0001), but not increases above and day 145. The PSPB profile from the goat-in-goat pregnancy basal concentrations (P > 0.05), were different for the different had a single peak at approximately day 60 and then steadily types of pregnancies. Females carrying ovine pregnancies (n = declined (Fig. 4d). 5) had higher absolute concentrations of PSPB at day 24 than

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access Table 3. Summary of mean pregnancy specific protein (PSPB) 1968; Roth et al, 1989), indicating slower growth. However, concentrations at day 24 concentrations of PSPB in the hybrid pregnancies were comparable to concentrations of PSPB in the one goat-in-goat established this Number of pregnancy during study. Placental formation, observed by did not Type of pregnancy animals PSPB (ng ml-1) ultrasonography, appear retarded in the hybrid-in-chimaera pregnancies when compared with sheep-in-sheep, sheep-in-chimaera and goat-in- Ovine conceptuses 5 12.69 + 1.86a goat pregnancies. pregnancies appeared to have 3 10.61 + 2-44 Hybrid-in-goat Sheep-in-sheep less developed placentomes at the same stage (days 40-57) 2 15.81 ± 0.70a Sheep-in-chimaera than those seen in hybrid-in-chimaera, conceptuses 6 4.13 ± 0.68a sheep-in-chimaera, Hybrid and and never 2 3.99 ± 0.86 sheep-in-sheep goat-in-goat pregnancies, pro¬ Hybrid-in-goat small the wall. These 4 4.20 ± 1.01a gressed beyond projections along uterine Hybrid-in-chimaera those Hancock Goat-in-goat 1 4.45 findings are consistent with of and McGovern (1970), who observed that the placenta in hybrid-in-goat preg¬ nancies failed to establish normal contact and were Values are means + SEM. placentomes however, there were also in aWithin a column, values with superscripts were significantly different underdeveloped; pregnancies (P < 0.025). which placentomes were normal in size but necrotic changes had taken place. McGovern (1973b) reported similar obser¬ cases did those carrying hybrid pregnancies (n = 6), regardless of the vations: cotyledons were in some inconspicuous and in abnormal maternal species (P < 0.025) (Table 3). Sheep-in-chimaera other cases well developed but in appearance. one doe PSPB concen¬ (n = 2) had PSPB concentrations than did In hybrid-in-goat pregnancy, 25, pregnancies higher of hybrid-in-chimaera pregnancies (n = 4) (P < 0.025). PSPB con¬ trations were exceptionally high, comparable to those intra¬ centrations were not different in sheep-in-sheep (n = 3) versus specific caprine pregnancies with multiple conceptuses (Humblot = the of more than one sheep-in-chimaera and hybrid-in-goat (n 2) versus hybrid-in- et al, 1990), although presence conceptus The concen¬ chimaera pregnancies (P > 0.05). When PSPB concentrations was never confirmed by ultrasonography. PSPB trations in this were also to those of chimaera on day 24 were corrected for basal values, concentrations in goat comparable that carried the sheep-in-chimaera pregnancies were higher than those for 8702, the sheep-hybrid chimaera hybrid preg¬ furthest that con¬ hybrid-in-chimaera pregnancies (P < 0.05). PSPB concen¬ nancy in gestation. This observation suggests centrations of PSPB are not useful for the health and trations after 30 days were highly variable and no relationships predicting between maternal species, type of pregnancy and concentration longevity of an interspecies pregnancy. lose second earlier than of PSPB were observed (P > 0.05). Does hybrid pregnancies in gestation they do the first (Bowerman and Hancock, 1963; McGovern, 1973a; MacLaren et al, 1992b), suggesting involvement of Discussion immune response in fetal death. Although sheep-goat chimaeras are tolerant to ovine and caprine species-specific antigens PSPB profiles of goat-in-goat pregnancies show a steady rise (MacLaren et al, 1992b; Gustafson et al, 1993), hybrid preg¬ reaching peak values at approximately day 121 with a sharp nancies were still lost. Loss occurred later in gestation than with decline after parturition (Humblot et al, 1990). The concen¬ sheep and goats carrying hybrid pregnancies, suggesting that tration of PSPB in the goat-in-goat pregnancy in this study they resulted from faulty fetal-maternal interactions. The reached a peak at approximately day 60 and then declined. sheep—hybrid chimaera carried the hybrid pregnancy the However, the concentrations of PSPB were comparable to those longest and had high PSPB concentrations with a profile similar of does carrying multiple conceptuses in the study of Humblot to that of a sheep-in-sheep pregnancy. The presence of hybrid et al. (1990). The PSPB profile of ewes is different from that of tissue in this animal may have contributed to appropriate fetal- does, showing two peaks at approximately day 60 and day 120 maternal interactions that allowed the pregnancy to be carried (Willard, 1991). Results from our sheep-in-sheep pregnancies for this long. The facial defects of the fetus suggest that the were consistent with this report. cause of pregnancy failure was a developmental problem. It is Hybrid pregnancies had lower concentrations of PSPB on unlikely that these deformities occurred because the fetus was day 24 than did ovine pregnancies. High basal PSPB concen¬ a hybrid; normal-looking hybrids have been recovered from trations in chimaeras that carried ovine pregnancies raise a hybrid-in-goat pregnancies (Warwick and Berry, 1949) and possibility that comparisons were influenced by unusually high occasional hybrids have developed to term (Bunch et al, 1976; concentrations during pregnancy in these two females. The Moore et al, 1981; Tucker et al, 1989). The genus Ovis has a reason for the high basal concentrations is unknown. The PSPB number of chromosomes varying from 54 to 58 (Nadler et al, antibody used in the radioimmunoassay crossreacts with ovine 1971); it is therefore believed that the chromosome number LH, prolactin, growth hormone and thyroid-stimulating hor¬ intermediate to the parental species of the hybrid is not mone in quantities greater than 1 pg per tube (Willard et al, responsible for its death in utero. Unexplained cranial defects 1987), but crossreactivity would not be expected to affect and hydranencephaly have been reported in Dall's sheep-in- analysis of sera from only two animals in the study. Low con¬ domestic sheep pregnancies (Buckerell et al, 1990). There was centrations of PSPB could be due to the smaller size of hybrid no evidence of hydranencephaly in the hybrid fetus recovered embryos and fetuses compared with sheep or goat fetuses at the in the case reported here and histological examination did not same stage (Warwick and Berry, 1949; Hancock and McGovern, reveal pathological organisms.

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access and Chimaera 8703 had a PSPB that could be considered Dunbar MM, Wong TS, Ruder-Montgomery CA, Chew BP Sasser RG (1990) profile Partial characterization of the of similar to a because there were two immunosuppressive properties pregnancy- sheep-in-sheep profile specific protein (PSPB) Theriogenology 33 220 (Abstract) the but there was corresponding peaks at appropriate times, Eckblad WP, Sasser RG, Ruder CA, Panlasigui PM and Kuczynski TS (1985) also a third peak (day 31) earlier than the first in sheep (day Localization of pregnancy-specific protein (PSPB) in bovine placental cells 60). Chimaera 8704 had a PSPB profile that rose, levelled using a glucose oxidase-anti-glucose oxidase immunohistochemical stain off and then declined by day 80 and remained low Proceedings of the Western Section American Society of Animal Science 36 throughout 396-397 These two different for the same of gestation. profiles type Fehilly CB and Willadsen SM (1986) Embryo manipulation in farm animals pregnancy (sheep-in-chimaera) could indicate influence of the Oxford Reviews of Reproductive Biology 8 379-413 maternal environment on the function of the binucleate cells Gustafson RA, Anderson GB, BonDurant RH and Mahi-Brown C (1993) Tolerance of chimaeras to their cells (the uterine chimaerism of these animals was unknown). sheep—goat component Journal of 23 155—168 Placental hormone may have been unusual in this Reproductive Immunology production Hancock JL and McGovern PT (1968) Transfer of goat x sheep hybrid eggs to animal as her cervix failed to dilate labour. Combined during sheep and reciprocal transfer of eggs between sheep and goats Research in with the unusual PSPB profile (reported here), progesterone Veterinary Science 9 411—415 profiles and placentas in other chimaeras (MacLaren et al, Hancock JL and McGovern PT (1970) Placentae of goat-sheep hybrids Journal of 1992a), maternal chimaerism to affect Anatomy 106 413 (Abstract) appears placental and Failure of of formation and function. Hancock JL, McGovern PT Stamp JT (1968) gestation goat x sheep hybrids in goats and sheep Journal of Reproduction and Fertility In summary, ultrasonography revealed that gross placental Supplement 3 29—36 development appeared more normal in hybrid-in-chimaera than Humblot P, De Montigny G, Jeanguyot N, Tetedoie F, Payen B, Thibier M and in this difference was not Sasser RG (1990) Pregnancy-specific protein and progesterone concen¬ hybrid-in-goat pregnancies, although trations in French reflected in PSPB concentrations. From 18 to 30, alpine goats throughout gestation Journal of Reproduction days hybrid and Fertility 89 205-212 had concentrations PSPB pregnancies lower peripheral of than McGovern PT (1973a) The effect of maternal immunity on the survival of did ovine pregnancies. If this difference is real, it could be a goat X sheep hybrid embryos Journal of Reproduction and Fertility 34 reflection of the smaller and possibly more slowly developing 215-220 McGovern PT (1973b) The fate of X embryos in hybrid fetus or fetal—maternal interactions affect¬ goat sheep hybrid goats compromised treated with semen Journal of and 34 of binucleate concen¬ parenterally sheep Reproduction Fertility ing function the cells. A decline in PSPB 221-225 trations was not clearly indicative of fetal death. The chimaeras MacLaren LA, Anderson GB, BonDurant RH and Edmondson AJ (1992a) Inter- carried hybrid pregnancies longer than did sheep and also for and intraspecific placentae in sheep, goats and sheep-goat chimaeras Journal longer than goats usually carry second hybrid pregnancies. of Comparative Pathology 106 279-297 Two chimaeras carried than MacLaren LA, Anderson GB, BonDurant RH, Edmondson AJ and Bernoco D hybrid pregnancies longer goats (1992b) Maternal serum to in first failure of reactivity species-specific antigens sheep—goat usually carry hybrid pregnancies, despite previous interspecific pregnancy Biology of Reproduction 46 1-9 caprine pregnancies and consequent exposure to caprine MacLaren LA, Anderson GB, BonDurant RH and Edmondson AJ Reproductive antigens. None of the hybrid pregnancies was carried to term. cycles and pregnancy in interspecific sheep—goat chimaeras Reproduction, Fertility and Development (in press) Moore NW, Hainan CRE, McKee JJ and Watson JI (1981) Studies on hybridiz¬ The authors thank B. Alexander, S. Donahue, A. Moyer and J. Rowe ation between a Barbary ram (Ammotragus lervia) and domestic ewes (Ovis for assistance with various aspects of this project and C. Mahi-Brown aries) and nanny goats (Capra hircus) Journal of Reproduction and Fertility 61 and D. Bernoco for helpful criticisms. P. Kennedy performed necropsy 79-82 of wild on the hybrid fetus recovered by Caesarian section. Funds were Nadler CF, Lay DM and Hassinger JD (1971) Cytogenetic analysis sheep provided by the USDA W-112 Regional Research Project. populations in northern Iran Cytogenetics 10 137-152 Reimers TJ, Sasser RG and Ruder CA (1985) Production of pregnancy-specific protein by bovine binucleate trophoblastic cells Proceedings of the Eighteenth Annual Meeting of the Society for the Study of Reproduction Abstract 55 Roth TL, Anderson GB, BonDurant RH and Pashen RL (1989) Survival of References sheep X goat hybrid inner cell masses after injection into ovine embryos Biology of Reproduction 41 675-682 Ruder CA, JN, Dahmen JJ and Sasser RG (1988) Detection of Allen WR (1975) The influence of fetal genotype upon endometrial cup Stellflug pregnancy of sera for and PMSG and in Journal of in sheep by radioimmunoassay pregnancy-specific protein development progestogen production equids 29 Reproduction and Fertility Supplement 23 405-413 Theriogenology 905-912 NA (1989) Placental Chimerism and its on Interspecies Pregnancy. PhD Anderson Bradford GE and PT of in ewes Ruffing Effect GB, Cupps (1981) Length gestation Thesis, of California, Davis lambs of two different breeds 16 119—129 University carrying Theriogenology SAS (1985) Statistical Analysis Systems (5th Edn). SAS Institute, Inc., Cary, NC Bowerman HRL and Hancock JL (1963) Journal of Sheep—goat hybrids Tucker EM, Denis and L (1989) Blood genetic marker studies of a and 6 i 26 Kilgour Reproduction Fertility sheep—goat hybrid and its back-cross offspring Animal Genetics 20 179—186 Buckrell BC, Gartley CJ, Mehren KG, Crawshaw GJ, Johnson WH, Barker IK, Warwick BL and Berry RO (1949) Inter-generic and intra-specific embryo Balke J, Coghill C, Challis JRG and Goodrowe KL (1990) Failure to maintain transfers in sheep and goats Journal of Heredity 40 297—303 interspecific pregnancy after transfer of Dall's sheep embryos to domestic Whitten WK and Biggers JD (1968) Complete culture of the preimplantation ewes and 90 387—394 Journal of Reproduction Fertility stages of the mouse in vitro Journal of Reproduction and Fertility 17 399—401 Bunch TD, Foote WC and Spillett JJ (1976) Sheep-goat hybrid karyotypes Willard JM (1991) Detection of Fetal Twins using a Specific Radioimmunoassay for Theriogenology 6 379-386 Pregnancy-Specific Protein B. MS Thesis, University of Idaho Butler JE, Hamilton WC, Sasser RG, Ruder CA, Hass GM and Williams RJ (1982) Willard JM, Ruder CA and Sasser RG (1987) Ovine pregnancy-specific protein Detection and partial characterization of two bovine pregnancy-specific concentrations in the sera of early pregnant and peripartum ewes Proceedings proteins Biology of Reproduction 26 925-933 of the Western Section of the American Society of Animal Science 38 231—233 Dent J, McGovern PT and Hancock JL (1971) Immunological implications of Wood AK, Darling A, Dusek GL, Sasser RG and Ruder CA (1986) Serum assay ultrastructural studies of goat X sheep hybrid placentae Nature 231 for detecting pregnancy in mule and white tailed deer Journal of Wildlife 116-117 Management 50 6S4-6&7

Downloaded from Bioscientifica.com at 10/06/2021 02:24:44AM via free access